1. Field of the Invention
The present invention is directed to a screening assay for antiviral compounds targeted to the HIV-1 gp41 core structure utilizing a conformation-specific monoclonal antibody, which is reactive with fusion active gp41 from human immunodeficiency virus type 1 (xe2x80x9cHIV-1xe2x80x9d) envelope glycoprotein. The present invention further relates to antiviral compounds targeted to the gp41 core structure of HIV-1.
2. Background Information
The infection of human immunodeficiency virus type I (HIV-1) is initiated by binding of the envelope glycoprotein (Env) surface subunit gp120 to both CD4 and particular chemokine receptors (i.e., CXCR4 and CCR5, etc.) on target cells. The Env transmembrane subunit gp41 concurrently dissociates from gp120 and then mediates the fusion of the viral and cellular membranes (Moore, J. P., B. A. Jameson, R. A. Weiss, and Q. J. Sattentau. 1993, xe2x80x9cThe HIV-cell fusion reactionxe2x80x9d, In Viral Fusion Mechanisms, J. Bentz, editors. CRC Press, Boca Raton. pp. 233-289; Berger, E. A. 1997, xe2x80x9cHIV-1 entry and tropism: the chemokine receptor connectionxe2x80x9d, AIDS, 11 (Supp. A): S3-16; Hunter, E., 1997, xe2x80x9cgp41, a multifunctional protein involved in HIV entry and pathogenesisxe2x80x9d, In Human Retroviruses and AIDS, 1997; Korber, B., Hahn, B., Foley, B., Mellors, J. W., Leitner, T., Myers, G., McCutchan, F., Kuiken, C. editors, Los Alamos National Laboratory, Los Alamos, N.M. p. 111-55-111-73; and Chan, D. C. and P. S. Kim, 1998, xe2x80x9cHIV entry and its inhibitionxe2x80x9d, Cell, 93, 681-684).
HIV-1 gp41 is composed of three domains, an extracellular domain (ectodomain), a transmembrane domain and an intracellular domain (endodomain). The gp41 ectodomain contains three major functional regions, i.e., the fusion peptide located at the N-terminus of gp41, followed by two 4-3 heptad repeats adjacent to the N- and C-terminal portions of the gp41 ectodomain, designated NHR (N-terminal heptad repeat) and CHR (C-terminal heptad repeat), respectively. The N- and C-terminal repeats were also named as xe2x80x9cHR1xe2x80x9d and xe2x80x9cHR2xe2x80x9d, respectively, by Rimsky, L. T., D. C. Shugars and T. J. Matthews, J. Virol., 72, 986-993.
Both NHR and CHR regions consist of hydrophobic amino acid sequences predicted to form xcex1-helices, denoted N and C helix (Caffrey, M., M. Cal, J. Kaufman, S. J. Stahl, P. T. Wingfield, D. G. Covell, A. M. Gronenborn, and G. M. Clore, 1998, xe2x80x9cThree-dimensional solution structure of the 44 kDa ectodomain of SIV gp41xe2x80x9d, EMBO J, 17, 4572-4584), which may function as essential structures required for oligomerization of gp41 and for conformational changes during the process of membrane fusion between HIV-1 and target cells (Gallaher, W. R., J. M. Ball, R. F. Garry, M. C. Griffin, and R. C. Montelaro, 1989, xe2x80x9cA general model for the transmembrane proteins of HIV and other retrovirusesxe2x80x9d, AIDS Res. Hum. Retroviruses, 5, 431-440; Delwart, E. L., G. Mosialos, and T. Gilmore, 1990, xe2x80x9cRetroviral envelope glycoprotein contain a leucine zipper-like repeatxe2x80x9d, AIDS Res. Hum. Retroviruses, 6, 703-706; Wild, C., T. Oas, C. McDanal, D. Bolognesi, and Matthews, T., 1992, xe2x80x9cA synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibitionxe2x80x9d, Proc. Natl. Acad. Sci. USA, 89, 10537-10541; Bernstein, H. B., S. P. Tucker, S. R. Kar, S. A. McPherson, D. T. McPherson, J. W. Dubay, J. Lebowitz, R. W. Compans, and E. Hunter, 1995, xe2x80x9cOligomerization of the hydrophobic heptad repeat of gp41xe2x80x9d, J. Virol., 69, 2745-2750).
Peptides derived from the NHR and CHR regions of gp41, designated N- and C-peptides (Chan, D. C, and P. S. Kim, 1998, xe2x80x9cHIV entry and its inhibitionxe2x80x9d, Cell, 93, 681-684), have potent antiviral activity against HIV-1 infection (Jiang, S., K. Lin, N. Strick, and A. R. Neurath, 1993, xe2x80x9cHIV-1 inhibition by a peptidexe2x80x9d, Nature, 365, 113; Wild, C. T., D. C. Shugars, T. K. Greenwell, C. B. McDanal, and T. J. Matthews, 1994, xe2x80x9cPeptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type I gp41 are potent inhibitors of virus infectionxe2x80x9d, Proc. Natl. Acad. Sci. USA, 91, 9770-9774; and Lu, M., S. C. Blacklow, and P. S. Kim, 1995, xe2x80x9cA trimeric structural domain of the HIV-1 transmembrane glycoproteinxe2x80x9d, Nat. Struct. Biol., 2, 1075-1082). Previous studies suggest that these peptides inhibit the membrane fusion step of HIV-1 infection, in a dominant-negative manner, by binding to viral gp41 (Chen, C. R, T. J. Matthews, C. B. McDanal, D. P. Bolognesi, and M. L. Greenberg, 1995, xe2x80x9cA molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusionxe2x80x9d, J. Virol., 69, 3771-3777; and Furuta, R., C. T. Wild, Y. Weng, and C. D. Weiss, 1998, xe2x80x9cCapture of an early fusion-active conformation of HIV-1 gp41xe2x80x9d, Nat. Struct. Biol., 5:276-279).
Limited proteolysis of a recombinant fragment of the gp41 ectodomain generated an N-peptide and a C-peptide, designated N-51 (spanning residues 540-590) and C-43 (residues 624-666). These two peptides overlap mostly the NHR and CHR regions. Several other N- and C-peptides (i.e., N-36, N-34, C-34 and C-28) were also produced (Lu, M. and P. S. Kim, 1997, xe2x80x9cA trimeric structural subdomain of the HIV-1 transmembrane glycoproteinxe2x80x9d, J. Biochem. Struct. Dynamic, 15:465-471). N- and C-peptides mixed at equimolar concentrations form stable xcex1-helical trimers of antiparallel heterodimers, representing the fusion-active (fusogenic) core domain of gp41. Crystallographic studies showed that this core domain is a six-stranded helical bundle. Three N helices associate to form the internal coiled-coil trimer via interaction of the residues at xe2x80x9caxe2x80x9d positions in the wheel of one N helix with those at xe2x80x9cdxe2x80x9d positions (see FIGS. 2 and 3) in that of another N helix. Three C helices pack obliquely against the outside grooves of the N helix trimer by the interaction of residues at xe2x80x9caxe2x80x9d and xe2x80x9cdxe2x80x9d positions in C helices with those at xe2x80x9cexe2x80x9d and xe2x80x9cgxe2x80x9d positions (see FIGS. 2 and 3) in N helices, respectively (Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim, 1997, xe2x80x9cCore structure of gp41 from the HIV envelope glycoproteinxe2x80x9d, Cell, 89, 263-273; Weissenhorn, W., A. Dessen, S. C, Harrison, I. I. Skehel, and D. C. Wiley, 1997, xe2x80x9cAtomic Structure of the Ectodomain from HIV-1 gp41xe2x80x9d, Nature, 387, 426-428; and Tan, K., I. Liu, I. Wang, S. Shen, and M. Liu, 1997, xe2x80x9cAtomic structure of a thermostable subdomain of HIV-1 gp41xe2x80x9d, Proc. Natl. Acad. Sci. USA, 94, 12303-12308).
The residues at these interaction sites are highly conserved and mutations of these residues may disrupt the six-stranded core structure and abolish HIV-1 infectivity (Cao, J., L. Bergeron, E. Helseth, M. Thali, H. Repke, and I. Sodroski, 1993, xe2x80x9cEffects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoproteinxe2x80x9d, J. Virol., 67, 2747-2755; Chen, S. S., C. N. Lee, W. R. Lee, K. Mcintosh, and T. H. Lee, 1993, xe2x80x9cMutational analysis of the leucine zipper-like motif of the human immunodeficiency virus type 1 envelope transmembrane glycoproteinxe2x80x9d, J. Virol., 67, 3615-3619; Wild, C., I. W. Dubay, T. Greenwell, T. Baird, Jr., I. G. Oas, C. McDanal, F. Hunter, and T. Matthews, 1994, xe2x80x9cPropensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complexxe2x80x9d, Proc. Natl. Acad. Sci. USA, 91, 12676-12680; Poumbourios, P., K. A. Wilson, R. I. Center, R. El Ahmar, and B. E. Kemp, 1997, xe2x80x9cHuman immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequencexe2x80x9d, J. Virol., 71, 2041-2049).
Each of the grooves on the surface of the N helices has a deep cavity that accommodates three conserved hydrophobic residues (W628, W631 and I635) in C helices. These highly conserved deep hydrophobic cavities have been suggested as attractive targets for development of antiviral lead compounds that block HIV-1-mediated membrane fusion and HIV-1 infection.
Several antiviral drugs targeted to HIV-1 reverse transcriptase (RT) and protease have been approved by the US Food and Drug Administration (FDA) in recent years for the treatment of HIV-1 infection and AIDS (Carpenter, C. C., M. A. Fischl, S. M. Hammer, M. S. Hirsch, D. M. Jacobsen, D. A., Katzenstein, J. S. Montaner, D. D. S., M. S. Richman, R. T. Schooley, M. A. Thompson, S. Vella, P. G. Yeni, and P. A. Volberding, 1998, xe2x80x9cAntiretroviral therapy for HIV infection in 1998: updated recommendations of the International AIDS Society USA Panelxe2x80x9d, JAMA, 280, 78-86). Combination therapy using these two types of inhibitors has been remarkably successful in reducing viral load and has lead to a decline in morbidity and mortality (Markowitz, M., M. Saag, W. G. Powderly, A. M. Hurley, A. Hsu, J. M. Valdes, D., Henry, F. Sattler, A. La Marca, Leonard J. M., and D. D. Ho, 1995, xe2x80x9cA preliminary study of ritonavir, an inhibitor of HIV-1 protease, to treat HIV-1 infectionxe2x80x9d, N. Engl. J. Med., 333, 1534-1539; Detels, R., A. Munoz, G. McFarlane, L. A. Kingsley, J. B. Margolick, J. Giorgi, L. X. Schrager, J. Phair, and for the Multicenter AIDS Cohort Study investigators, 1998,xe2x80x9d xe2x80x9cEffectiveness of potent antiretroviral therapy on time to AIDS and death in men with known HIV infection durationxe2x80x9d, JAMA, 280, 1497-1503; Hogg, R. S., S. A. Rhone, B. Yip, C. Sherlock, B. Conway, M. T. Schechter, M. V. O""Shaughnessy, and J. S. Montaner, 1998, xe2x80x9cAntiviral effect of double and triple drug combinations amongst HIV-infected adults: lessons from the implementation of viral load-driven antiretroviral therapyxe2x80x9d, AIDS, 12, 279-284; Hogg, R. S., K. V. Heath, B. Yip, K. J. Craib, M. V. O""Shaughnessy, M. T. Schechter, and I. S. Montaner, 1998, xe2x80x9cImproved survival among HIV-infected individuals following initiation of antiretroviral therapyxe2x80x9d, JAMA, 279, 450-454; Palella, F. J.Jr., K. M. Delaney, A. C. Moorman, M. O. Loveless, J. Fuhrer, G. A. Satten, D. J. Aschman, and S. D. Holmberg, 1998, xe2x80x9cDeclining morbidity and mortality among patients with advanced human immunodeficiency virus infection, HIV Outpatient Study Investigatorsxe2x80x9d, N. Eng. J. Med., 338, 853-860). However, these drugs have a number of shortcomings, namely, (1) the emergence of HIV-1 mutant strains having single or multiple resistance to the drugs used (Gunthard, H. F., J. K. Wong, C. C. Ignacio, J. C. Guatelli, N. L. Riggs, D. V. Havlir, and D. D. Richman, 1998, xe2x80x9cHuman immunodeficiency virus replication and genotypic resistance in blood and lymph nodes after a year of potent antiretroviral therapyxe2x80x9d, J. Virol., 72, 2422-2428; Richman, D. D., 1996, xe2x80x9cAntiretroviral drug resistance: mechanisms, pathogenesis, clinical significancexe2x80x9d, Adv. Exp. Med. Biol., 394, 383-395; Wong, J. K., H. F. Gunthard, D. V. Havlir, Z. Q. Zhang, A. T. Haase, C. C. Ignacio, S. Kwok, E. Emini, and D. D. Richman, 1997, xe2x80x9cReduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failurexe2x80x9d, Proc. Natl. Acad. Sci. USA, 94, 12574-12579); (2) Adverse side effects; and (3) high cost (Montaner, J. S., R. S. Hogg, A. E. Weber, A. H. Anis, M-V. O""Shaughnessy, and M. T. Schechter, 1998, xe2x80x9cThe costs of triple-drug anti-HIV therapy for adults in the Americasxe2x80x9d, JAMA, 279, 1263-1264). In addition, these drugs are targeted to later stages of infection. Therefore, it is essential to develop compounds with higher effectiveness and lower side effects which can prevent early steps of HIV-1 infection.
The C-peptides block in vitro HIV-1 infection and cell fusion at nM concentrations. In a recent phase I/II clinical trial in humans, T-20, one of the C-peptides showed potent in vivo inhibition of HIV-1 replication, resulting in viral load reduction comparable to that obtained by the 3-4 combination therapies with RT and protease inhibitors (Kilby, J. M., S. Hopkins, T. M. Venetta, B. DiMassimo, G. A. Cloud, J. Y. Lee, L. Alldredge, E. Hunter, I. D. Lambert, D. Bolognesi, T. Matthews, M. R. Johnson, M. A. Nowak, G. M. Shaw, and M. S. Saag, 1998, xe2x80x9cPotent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entryxe2x80x9d, Nature Med., 4, 1302-1367). Despite this early success, the application of this peptide therapy may be limited due to the high production cost of the peptide and lack of its oral availability. Thus, identification of small molecule inhibitors reacting with the same or overlapping target sites on gp41 recognizing the antiviral peptides needed.
U.S. Pat. No. 5,840,843 describes a synthetic HIV-1 polypeptide which comprises an amino acid sequence corresponding to the amino acid sequence of the envelope glycoprotein of HIV-1IIIB virus from the amino acid residue 600 to the amino acid residue 862. U.S. Pat. No. 5,840,843 refers to xe2x80x9cpeptide (637-666)xe2x80x9d, based on its sequence in gp41 (amino acid residues 637-666). The sequence was numbered according to Ratner""s publication (Nature, 313, 227-284, 1985). However, most AIDS researchers later numbered the gp41 sequence according to a book entitled xe2x80x9cHuman Retrovirus and AIDSxe2x80x9d. According to this book, the sequence of xe2x80x9cpeptide (637-666)xe2x80x9d was renumbered as xe2x80x9c630-659xe2x80x9d and the peptide was renamed as xe2x80x9cSJ-2176xe2x80x9d. This peptide has 30 residues overlapping the peptide xe2x80x9cC34xe2x80x9d (628-661).
EP 335,134 describes a mouse monoclonal antibody to human immunodeficiency virus gp41 protein.
U.S. Pat. No. 5,777,074 and EP 492,560 describe a method for neutralizing HIV-1 utilizing a human monoclonal antibody directed against the transmembrane glycoprotein (gp41) of HIV-1 (also see U.S. Pat. No. 5,459,060).
U.S. Pat. No. 5,166,050 describes monoclonal antibodies and peptides useful in treating and diagnosing HIV infections.
It is an object of the present invention to provide methods for the screening of antiviral compounds targeted to the HIV-1 gp41 core structure.
It is also an object of the present invention to provide a conformation-specific monoclonal antibody which reacts with fusion-active gp41 from the HIV-1 envelope glycoprotein.
It is a further object of the present invention to provide a screening assay for antiviral compounds targeted to the HIV-1 gp41 core structure using the conformation-specific monoclonal antibody which reacts with fusion-active gp41 from the HIV-1 envelope glycoprotein.
It is a still further object of the present invention to provide compounds which are effective against HIV-1 infection.
It is another object of the present invention to provide methods for inhibiting HIV-1 virus replication or infectivity or treating HIV-1 infection in a subject without inducing undesirable immunosuppressive effects.
The above objects, as well as other objects, aims and advantages, are satisfied by the present invention.
The present invention concerns methods for the screening of antiviral compounds targeted to the HIV-1 gp41 core structure using (i) polyclonal and monoclonal antibodies, and only (ii) monoclonal antibodies.
A first method for the screening of antiviral compounds targeted to the HIV-1 gp41 core structure, which involves the use of polyclonal and monoclonal antibodies, comprises:
(a) capturing polyclonal antibodies from an animal other than a mouse, directed against a trimer of a heterodimer containing an N-peptide and a C-peptide, onto a solid-phase to form a polyclonal antibody coated solid-phase;
(b) mixing a compound to be tested with an N-peptide, and then adding a C-peptide thereto;
(c) adding the mixture from step (b) to the polyclonal antibody coated solid-phase form step (a), and then removing unbound peptides and unbound compound;
(d) adding a monoclonal antibody directed against the trimer of a heterodimer containing an N-peptide and a C-peptide, and
(e) measuring the binding of the monoclonal antibody (for example, by sequentially adding biotin labeled anti-mouse IgG, streptavidin or avidin labeledenzyme, and a substrate for generating detectable color).
A second method for the screening of antiviral compounds targeted to the HIV-1 gp41 core structure, which involves the use of monoclonal antibodies, comprises:
(a) capturing a C-peptide onto a solid-phase to form a C-peptide coated solid-phase;
(b) mixing a compound to be tested with an N-peptide;
(c) adding the mixture from step (b) to the C-peptide coated solid-phase from step (a), and then removing unbound peptide and unbound compound;
(d) adding a monoclonal antibody directed against a trimer of a heterodimer containing an N-peptide and a C-peptide, and
(e) measuring the binding of the monoclonal antibody (for example, by sequentially adding biotin labeled anti-mouse IgG, streptavidin or avidin labeled enzyme, and a substrate for generating detectable color).
A third-method for the screening of antiviral compounds targeted to the HIV-1 gp41 core structure, which involves the use of monoclonal antibodies, comprises:
(a) capturing an N-peptide onto a solid-phase to form an N-peptide coated solid-phase;
(b) mixing a compound to be tested with a C-peptide;
(c) adding the mixture from step (b) to the N-peptide coated solid-phase from step (a), and then removing unbound peptide and unbound compound;
(d) adding a monoclonal antibody directed against a trimer of heterodimer containing an N-peptide and a C-peptide, and
(e) measuring the binding of the monoclonal antibody (for example, by sequentially adding biotin labeled anti-mouse IgG, streptavidin or avidin labeled enzyme, and a substrate for generating detectable color).
The present invention also concerns a monoclonal antibody which reacts with the fusion-active gp41 core structure and which binds specifically to a trimer of a heterodimer formed by an N-peptide and a C-peptide, but not to the individual N-peptide and C-peptide. The present invention is further directed to a conformation-specific monoclonal antibody which binds specifically to the oligomeric forms of gp41 and to the surfaces of HIV-1 infected cells only in the presence of soluble CD4.
The present invention also relates to a method for the screening of antiviral compounds targeted to the HIV-1 gp41 core structure by utilizing the conformation-specific monoclonal antibody described herein in an assay, such as an enzyme-linked immunosorbent assay (ELISA).
The present invention is further directed to a method of inhibiting HIV-1 virus replication or infectivity in cells comprising contacting the cells with a compound selected from the group consisting of 7-[6-phenylamino-4-[4-[(3,5-disulfo-8-hydroxynaphthyl)azo]-2-methoxy-5-methylphenylamino]-1,3,5,triazine-2-yl]-4-hydroxy-3-[(2-methoxy-5-sulfophenyl)azo]-2-naphthalene sulfonic acid and 5-[(4-chloro-6-phenylamino-1,3,5-triazine-2-yl)-amino]-4-hydroxy-3-[(4-methyl-6-sulfophenyl)azo]-2,7-naphthalene disulfonic acid.
The present invention also concerns a method of inhibiting HIV-1 virus replication or infectivity in a patient or for treating a patient infected with HIV-1 comprising administering to the patient an effective anti-HIV-1 amount of at least one pharmaceutically active compound selected from the group consisting of 7-[6-phenylamino-4-[4-[(3,5-disulfo-8-hydroxynaphthyl)azo]-2-methoxy-5-methylphenylamino]-1,3,5,triazine-2-yl]-4-hydroxy-3-[(2-methoxy-5-sulfophenyl)azo]-2-naphthalene sulfonic acid and 5-[(4-chloro-6-phenylamino-1,3,5-triazine-2-yl)-amino]-4-hydroxy-3-[(4-methyl-6-sulfophenyl)azo]-2,7-naphthalene disulfonic acid, alone, or in combination with a pharmaceutically acceptable carrier.